Abstract
Eukaryotic RNA polymerase II transcribes all protein-coding mRNAs and is highly regulated. A key mechanism directing RNA polymerase II and facilitating the co-transcriptional processing of mRNAs is the phosphorylation of its highly repetitive carboxyl-terminal domain (CTD) of its largest subunit, RPB1, at specific residues. A variety of techniques exist to identify and quantify the degree of CTD phosphorylation, including phosphorylation-specific antibodies and mass spectrometry. Electrophoretic mobility shift assays (EMSAs) have been utilized since the discovery of CTD phosphorylation and continue to represent a simple, direct, and widely applicable approach for qualitatively monitoring CTD phosphorylation. We present a standardized method for EMSA analysis of recombinant GST-CTD substrates phosphorylated by a variety of CTD kinases. Strategies to analyze samples under both denatured/reduced and semi-native conditions are provided. This method represents a simple, direct, and reproducible means to monitor CTD phosphorylation in recombinant substrates utilizing equipment common to molecular biology labs and readily applicable to downstream analyses including immunoblotting and mass spectrometry.
Highlights
[Background] Eukaryotic RNA polymerase II (RNAPII) generates all protein-coding mRNAs, small nuclear, small nucleolar, and many micro RNAs (Jeronimo et al, 2013; Mayfield et al, 2016)
The carboxyl-terminal domain (CTD) undergoes dephosphorylation and is recycled to initiate another round of transcription. This is achieved through the action of multiple CTD kinases; including CDK7, of the TFIIH complex, and CDK9, of the Positive transcription elongation factor b (P-TEFb) complex; and their counterparts the CTD phosphatases, including SSU72 and CTDP1 (Jeronimo et al, 2013; Mayfield et al, 2016)
Distinct pools of unphosphorylated and hyperphosphorylated RPB1 are detected in cell lysates using polyacrylamide gel electrophoresis (PAGE) due to a dramatic shift in the isoelectric point of RPB1 resulting from hyperphosphorylation
Summary
GST fusions of the CTDs from various organisms, including Homo sapiens, Drosophila melanogaster, and Saccharomyces cerevisiae, are utilized extensively. Elute GST-yeast CTD from the column by adding 10 ml of Elution buffer and allowing the solution to slowly drip from the chromatography column and collect it in a clean 50 ml conical tube. Pool fractions containing GST-yeast CTD and concentrate in Amicon centrifugal unit until approximately 10mg/ml as determined by absorbance at 280 nm on NanoDrop. Kinase reactions Combine stock solutions as follows: 5 μl 4x Kinase Buffer (Recipe 8) 5 μl 4x GST-yeast CTD Substrate (Recipe 10) 5 μl 4x Kinase Solution (Recipes 11, 12, 13 as desired) 5 μl 4x ATP Initiate reactions by adding 4x ATP. 2. No kinase control reactions Combine stock solutions as follows: 5 μl 4x Kinase Buffer (Recipe 8) 5 μl 4x GST-yeast CTD Substrate (Recipe 10) 5 μl Deionized water 5 μl 4x ATP Initiate reactions by adding 4x ATP. Completed reactions can be analyzed immediately or stored at -80 °C for up to 1 year
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